Method for analyzing proteins

ABSTRACT

A method for analyzing proteins makes use of an array of first capture molecules which are specific for peptide epitopes. The proteins to be analyzed or a protein mixture containing the proteins to be analyzed is degraded to peptide fragments corresponding to the peptide epitopes, after which the array of capture molecules is incubated with the peptide fragments. The peptide fragments bound to the capture molecules are then detected.

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation of the International PatentApplication PCT/EP01/12295 filed on Oct. 24, 2001, designating the U.S.,and published in German, which claims priority to German PatentApplication DE 100 54 055.4, filed on Oct. 31, 2000, both of which areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method for analyzing proteinsin which an array of first capture molecules which are specific forpeptide epitopes is employed, and to a corresponding capture molecule.

[0004] 2. Description of the Related Art

[0005] Numerous methods of this type are known from the prior art, andthey are used for the qualitative or quantitative analysis of proteins.

[0006] Examples of the areas of use of the known methods are proteinanalysis and protein detection. A further novel area of application ofthe known methods is the area of proteomics, a branch of researchdevoted entirely to proteins. One aspect of proteomics deals withcomparing the protein composition in pathologically altered cellscontrasted with normal, healthy cells. Investigations of this type areat present usually carried out by two-dimensional gel electrophoresis.Another field investigates the spatial structure of proteins, which isof interest in particular for drugs companies for developing novelmedicaments.

[0007] In two-dimensional gel electrophoresis, the protein mixture to beanalyzed is applied to a solid support, and a first separation takesplace according to the content of acidic and basic amino acid units inthe proteins via a pH gradient. The proteins are then fractionated inthe second direction by an electric field, to result in a pattern ofspots in which each spot represents one protein. It is possible bycomparing the patterns of spots to analyze differences in proteincompositions between healthy and pathologically altered cells. Foridentification, the protein spots are cut out and the proteins arefragmented by digestion with specific proteases. The masses of thefragments remaining after this treatment are characteristic of eachprotein.

[0008] In the method mentioned in the introduction for analyzingproteins, the detection takes place, for example, with protein-specificantibodies in array format.

[0009] To obtain such protein-specific antibodies, the applicant employsisolated, purified proteins, recombinantly produced proteins orchemically synthesized peptides derived from the protein sequence. Theantibodies are obtained by immunization of laboratory animals with theproteins or peptides acting as antigens, and with adjuvants. It is alsopossible on the other hand to obtain the antibodies by in vitro methodsas recombinant antibodies.

[0010] The peptides which are to be synthesized chemically are derivedfrom known protein sequences, which are present, for example, in proteindatabases or can be found from nucleic acid databases, using softwareprograms which allow theoretical predictions to be made about theprotein structure and a possible antigenicity. Programs of this type aredescribed, for example, by Lars Hennig: “WinPep—ein Programm zur Analysevon Aminosäuresequenzen”, BIOSPEKTRUM 4 (5), 1998, pages 49-50; or byDevereux et al.: “A Comprehensive Set of Sequence Analysis Programs forthe VAX”, NUCLEIC ACIDS RESEARCH, volume 12, 1984, pages 387-395. Withthese programs it is possible to list all possible fragments, withmolecular weight, sequence, sequence position and length, which aregenerated on cleavage of proteins for example by proteases or chemicalagents. To predict the antigenicity, the probability with which theepitope is located on the surface of the protein is found on the basisof structural predictions; see, for example, Hopp and Woods: “A computerprogram for predicting protein antigenic determinants”, Mol. Immunol.,volume 20, 1983, pages 483-489; further literature references are to befound in the worldwide web under the address www.expasy.ch.

[0011] Although the antibodies generated through application of thesetechniques are very good at recognizing the peptide employed forimmunization, they often do not bind to the corresponding peptideepitope in the native protein. The reason for this may be, for example,that the peptide epitope in the native protein is not accessible to theantibody, for steric reasons, or that it is present, as a result ofpost-translational modification, in a modified form which cannot bederived from the database. A further reason may be that the peptideepitope is present in the native protein in a conformation whichprevents antibody binding.

[0012] Thus, a disadvantage of this technique is the initial elaborategeneration of a large number of peptide-specific antibodies againstprotein epitopes, which subsequently bind only poorly or not at all tothe protein which is sought.

[0013] Proteins can be detected using such protein-specific antibodies,on the one hand, after fractionation by means of SDS-PAGE and transferto membranes by Western blotting/immunoblotting techniques or by meansof the ELISA technique. In the ELISA technique, the protein to bedetected is bound, without previous separation of other proteins from acomplete protein solution, directly,to a highly specific antibody whichis immobilized on a solid support. The protein solution is then washedoff the solid support, and the protein which is bound, i.e. remaining onthe support, is detected.

[0014] This detection takes place either with the aid of labeled secondantibodies which are specific for the bound protein, or by competitionof the analyte protein with labeled analyte protein which is added tothe solution in defined amounts. This indirect detection makes use ofthe fact that unlabeled analyte protein from the sample and addedlabeled analyte protein compete in a manner which is defined by the lawof mass action for the binding sites which are present. This results inthe amount of bound, labeled analyte protein being inverselyproportional to the amount of unlabeled analyte protein in the sample.

[0015] This method has on the one hand the previously mentioneddisadvantage, namely that the preparation of the specific antibodiesleads to a large number of antibodies which bind only poorly or not atall to the protein to be analyzed. A further disadvantage on use of theWestern blotting technique is that the protein mixture fractionated bySDS-PAGE can in each case be tested only with one antibody or withdifferently labeled antibodies, respectively, in order to ensuredistinguishability of the antibodies bound to the different proteins.

[0016] In the ELISA technique it is also always possible for only oneantibody to be immobilized per analysis well and then incubated with theprotein solution. This technique requires large amounts of samplebecause each analysis well must be incubated with one aliquot of theprotein mixture. A further disadvantage in the known methods is thatdetection of the bound proteins requires specific second antibodies foreach protein to be detected or large amounts of labeled analyte proteinsfor competition experiments.

[0017] As already mentioned in the introduction, the applicant alsofunctionally immobilizes protein-specific antibodies differing inspecificity in array format in rows and columns on a support material.The proteins to be analyzed are labeled and then incubated with theantibodies on the array. The proteins in the solution which are antigensfor the immobilized antibodies then bind to the antibodies which arespecific for them, resulting in spatially resolved specific proteinbinding.

[0018] It is possible on the basis of the known binding specificities ofthe immobilized antibodies and of the known positions of the respectiveantibodies in the array to determine the bound amount of the respectiveproteins in parallel. For this purpose, the bound amount of protein ismeasured via the labeling on the proteins by means of a spatiallyresolved detection.

[0019] Besides qualitative parallel detection of different proteinspresent in the sample solution, it is in addition to that possible byuse of standard proteins to determine quantitatively the analyteproteins.

[0020] In contrast to the method described above, the advantage here isthat a plurality of proteins can be detected in parallel in the samesample and in one well. However, the disadvantage is that the proteinsto be analyzed must be labeled, for which purpose appropriate labelsmust be introduced on particular functional groups of individual aminoacids of the proteins. The efficiency of such labeling reactions varieswidely for different proteins, and it is determined by the particularimmediate microenvironment of a functional group, i.e. by stericshielding, pH variation in the direct vicinity of the functional groupdue to neighboring groups, salts, solvents etc. In relation to theresult, this means that the reactivity of chemically identicalfunctional groups in a protein may vary widely, so that quantitativereaction of certain functional groups is very difficult.

[0021] In addition, the labeling reaction may lead to modification ofamino acids within an epitope recognized by the specific antibody, whichleads to loss of binding between protein epitope and antibody. However,this means that the protein is no longer detectable via the describedassay method after the labeling.

SUMMARY OF THE INVENTION

[0022] Against this background, an object underlying the presentinvention is to improve the method mentioned at the outset such thatquantitative and/or qualitative analysis of proteins becomes possible ina simple, rapid and reliable way.

[0023] According to the invention this object is achieved with themethod comprising the steps:

[0024] degradation of the proteins to be analyzed or of a proteinmixture containing the proteins to be analyzed to peptide fragmentscorresponding to the peptide epitopes,

[0025] incubation of the array with the peptide fragments, and

[0026] detection of peptide fragments bound to the first capturemolecules.

[0027] This object on which the invention is based is completelyachieved in this way. The invention is based on the surprisingrealization by the inventors that the capture molecules immobilized inarray format are incubated not as in the prior art with the proteins butafter, for example, enzymatic cleavage of the proteins with individualpeptide fragments which correspond to the peptide epitopes which areemployed for generating the specific first capture molecules.

[0028] One advantage of this method is that the secondary and tertiarystructures of the proteins to be analyzed are irrelevant, and the firstcapture molecules recognize the corresponding peptide fragments withgreat certainty because they were generated against correspondingpeptide epitopes. These epitopes can be predicted directly fromdatabases, and additional elaborate prediction of secondary and tertiarystructures is unnecessary. The novel method is not only very reliable inthis way, it is also simple and quick to carry out because the firstcapture molecules reliably recognize the proteins via the detectedpeptide fragments.

[0029] A further advantage is to be seen in the fact that each capturemolecule recognizes its optimal antigen, since the synthetic peptideused to generate capture molecules is identical to the peptide fragmentto be detected. The disadvantage present in the prior art is avoided inthis way, namely that it was up to now not possible with proteins topredict whether a peptide-specific antibody also recognizes the nativeprotein. In other words, this means that with the first capturemolecules there is a distinct reduction in the rejected materialcompared with the prior art.

[0030] Since the arrangement of the different first capture molecules inthe array is known, it is possible by means of an automatable, spatiallyresolved detection to identify qualitatively and determinequantitatively the fragments and thus the proteins. This is because thepeptide patterns found correlate directly with the protein patterndefined as analyte protein on designing the array. It is additionallypossible for peptide patterns found to be screened against DNA databasesusing simple, fast algorithms.

[0031] It is possible in this way to obtain information quickly andreliably about which proteins are present in a sample solution, whatmethod can be employed for example in diagnostics. In addition, thenovel method makes it possible for the first time to carry out simpleanalysis of mutations at the protein level.

[0032] A further advantage of the known method is its speed, since thelow molecular weight fragments permit binding assays to be carried outfaster than do higher molecular weight proteins. The result is thus akinetic speeding up of the assays, and similar kietics of analytebinding lead to simpler establishment of optimal conditions for theassays than is the case with the highly variable kinetics resulting withanalyte proteins which vary greatly in their molecular weight.

[0033] A further advantage of the novel method is that a defined andcomplete degradation of the proteins to be analyzed to give the peptidefragments is possible, and that, in contrast to proteins, the fragmentscan be quantitatively labeled on defined functional groups. This leadsto detection of the peptide fragments being possible not onlyqualitatively but also quantitatively without needing to take account ofsecondary and tertiary structures.

[0034] A further object is the method according to the invention,wherein fragments which are unbound are washed away after theincubation.

[0035] The advantage of this is that the specificity of the method isincreased in a manner known per se.

[0036] A further object is the method according to the invention,wherein the peptide fragments are labeled before the incubation.

[0037] Since a specific, complete labeling of the peptide fragments ondefined functional groups is possible, quantitative determination ofeach bound peptide epitope can be carried out in this way. It isadvantageous in this connection that complete labeling of the peptidefragments is more easily and more reproducibly possible than labeling ofproteins as carried out in the prior art.

[0038] A further object is the method according to the invention,wherein the detection takes place via labeled second capture moleculeswhich specifically recognize the peptide fragment bound to the firstcapture molecule.

[0039] It is advantageous here on the one hand that the peptidefragments need not be labeled, so that the method can be carried outoverall more quickly, since labeled second capture molecules can beprovided in large quantity in a single process step, after which it isthen possible to carry out many analytical methods of the invention withdifferent samples of proteins, each of which needs to be degraded onlyto the corresponding peptide fragments.

[0040] A further advantage of this measure is that the selectivity isincreased, since peptide fragments nonspecifically bound by the firstcapture molecule are not recognized because the second capture moleculesdo not bind to these complexes.

[0041] A further object is the method according to the invention,wherein the second capture molecules are generated starting fromcomplexes of first capture molecules and peptide epitopes bound thereto.

[0042] The advantage of this is that very specific second capturemolecules are generated and can, moreover, be prepared with a highsuccess rate.

[0043] A further object is the method according to the invention,wherein the first capture molecules are generated starting from peptideepitopes which are prepared well-aimed in relation to peptide fragmentsresulted from degradation of the proteins to be analyzed.

[0044] This measure is associated with a whole series of advantages. Onthe one hand, the first capture molecules against the linear peptideepitopes can be produced with a high success rate, because the peptideepitopes for preparing and isolating the capture molecules arecompletely identical to the peptide epitopes to be analyzed. This leadsto considerably less rejected material being produced in the preparationof the first capture molecules than in the preparation ofprotein-specific capture molecules. Such peptide epitopes for preparingand isolating the capture molecules can be prepared and completelycharacterized analytically quickly and at low cost, which represents afurther advantage compared with the prior art.

[0045] In addition, this measure also increases the specificity of thenovel method. This is because epitopes occurring in different proteinscan be supplemented by further protein-specific epitopes in such a waythat the combination of epitopes detected on the array is unambiguousfor particular analyte proteins. Not only is there an increase in thespecificity and redundancy of the novel method in this way, targetedoptimization for detecting particular proteins is also possible.

[0046] In this connection, the peptide epitopes can be prepared bychemical synthesis or enzymatic degradation from known proteins, so thatinexpensive and fast complete analytical characterization thereof ispossible with standard methods.

[0047] A further object is the method according to the invention,wherein the well-aimed prepared peptide epitopes are selected frompotential peptide epitopes of the proteins to be analyzed.

[0048] One advantage in this case is that only peptide epitopes areprepared against which capture molecules can also be generated. Thismeasure thus leads to a saving of time and synthesis for the preparationof the first and/or second capture molecules. “Potential” peptideepitopes mean in this connection all possible epitopes of a protein,i.e. including those which are not present on the surface of the proteinand have not to date been used for generating capture molecules. Afurther advantage in this connection is that many more possible epitopescan be considered for each protein than in the prior art, so that, forexample in the case of very similar or closely related proteins, a muchlarger number of peptide epitopes is available for possibledifferentiation between the proteins.

[0049] A further advantage is that only peptide epitopes are preparedwhich are indeed specific for a protein or for a few proteins.

[0050] A further object is the method according to the invention,wherein the potential peptide epitopes are derived from known amino acidand/or nucleic acid sequences of the proteins to be analyzed.

[0051] One advantage in this case is that the peptide epitopes ofproteins to be detected are predicted from DNA or protein databaseswithout taking account of the tertiary structure of the proteins. Asimple prediction of the protein cleavage sites for the agents employedfor a specific protein cleavage is sufficient, a selection being madefrom one or more peptide epitopes with maximal specificity for theanalyte protein. In the optimal case, the epitope occurs only in theanalyte protein.

[0052] A further object is the method according to the invention,wherein potential peptide epitopes are selected from sequence sectionswhich are retained on degradation of the proteins to be analyzed.

[0053] With this measure, the saving of time is advantageous becauseonly capture molecules are generated for which peptide fragments canalso be produced, care being taken with the potential peptide epitopesthat they are specific for one or for a few analyte proteins and thattheir preparation is possible with high probability.

[0054] Prediction of peptide sequences which are difficult to obtain inchemical synthesis can be employed as further criterion for selectingthe peptide epitopes. Conventional prediction of immunogenic linearpeptide sequences, in which potential linear peptide epitopes areidentified in protein structures on the basis of the hydropathicprofiles (Kyte and Doolittle, “A Simple Method for Displaying theHydropathic Character of a Protein”, J. MOL. BIOL. 157 (1982), pages105-132) and prediction of β-loop-inducing sequence sections (Chou andFasman, “Prediction of Protein Conformation”, BIOCHEMISTRY 13, 1974,pages 222-245), is unnecessary in this case because, after denaturationand degradation of the analyte proteins, all peptide fragments representpotential peptide epitopes, irrespective of their position in theprotein structure.

[0055] This leads to a larger choice of potential peptide epitopes andthus to a greater probability of finding epitopes specific for proteinsto be analyzed (hereinafter: analyte proteins) than with arrays ofcapture molecules which are incubated with complete analyte proteins.

[0056] A further object is the method according to the invention,wherein the well-aimed prepared peptide epitopes are labeled and/ormodified like the peptide fragments after/during the degradation of theproteins to be analyzed.

[0057] One advantage of this measure is that it leads to a highspecificity of the first capture molecules for the peptide fragments,because account is taken even during the preparation of the peptideepitopes used to generate the first capture molecules of whether thepeptide fragments are labeled or modified after/during the degradationof the proteins to be analyzed.

[0058] A further advantage is to be seen in the well-aimed incorporationof labels in automated chemical synthesis, thus making rapid andlow-cost, completely defined labeling possible, which is simpler, lesscostly and more reproducible than labeling of complete protein.

[0059] A further object is the method according to the invention,wherein the well-aimed prepared peptide epitopes are provided withpost-translational modifications.

[0060] An advantage of this procedure is that the proteins to beanalyzed can be screened specifically for post-translationalmodifications. It is moreover possible to prepare, for example, peptideepitopes from the region of potential phosphorylation sites of a proteinas synthetic phosphopeptides and as nonphosphorylated peptides, in orderto generate capture molecules for the respective peptide epitopes. Anarray of such capture molecules which recognize the differentphosphorylated and nonphosphorylated peptide epitopes can then beemployed to quantify states of phosphorylation of one or more proteinsdirectly from a protein mixture. Other modifications which can bescreened in this way include modified amino acids, glycopeptides, etc.

[0061] A further object is the method according to the invention,wherein the well-aimed prepared peptide epitopes have at least one aminoacid exchange and/or at least one deletion of an amino acid comparedwith the potential peptide epitopes.

[0062] An advantage of this measure is that a single nucleotidepolymorphism can be identified, since the exchange or the deletion,respectively, of individual amino acids in proteins can be detected andquantified in this way. For this purpose, epitopes from the region ofthe amino acid exchange are prepared as synthetic peptides in order togenerate specific capture molecules for these epitopes. After enzymaticdegradation of the corresponding proteins and, if appropriate, labelingof the generated peptide fragments, and incubation with the array, thevarious peptides possible through the single nucleotide polymorphism andhaving the different amino acid exchanges and deletions can be detectedand quantified directly on the array.

[0063] A further object is the method according to the invention,wherein the or each capture molecule to be an antibody, an antibodyfragment, a peptide aptamer or another capture molecule which can beprepared recombinantly by mutation of binding domains or chemicalsynthesis.

[0064] A further object is the method according to the invention,wherein the binding specificity of the capture molecules to beestablished by binding assays using peptides which have been altered inindividual amino acids or in their length. This makes it possible forthe specificity of the generated capture molecules to be characterizedcompletely, and rapidly and inexpensively, to avoid cross-reactivitywith other epitopes. With proteins themselves this cannot be carried outwith reasonable effort.

[0065] This can now take place according to the invention in the arraywith synthetic peptides each of a defined sequence or with peptidelibraries against individual immobilized capture molecules andsubsequent identification of bound peptides.

[0066] Against this background, another object of the present inventionis a capture molecule, in particular for use in a method of the abovetype, which binds specifically to peptide epitopes which correspond topeptide fragments into which the proteins to be analyzed can bedegraded, where the peptide epitopes in the protein itself arepreferably not accessible to the capture molecule.

[0067] With these capture molecules which have not been discoverable inthe prior art to date, it is advantageous that they are able veryspecifically also to distinguish very similar or closely relatedproteins, because the number of peptide epitopes which are not presenton the surface, and which are thus for example masked in the proteinitself, is very large. In this way, the number of available peptideepitopes which have not been taken into account in the prior art to datefor generating specific antibodies which can be employed fordiagnostics, mutation analysis etc. at the protein level is very large.

[0068] A further object of the present invention is a capture moleculewhich specifically binds to a complex of an aforementioned capturemolecule and the corresponding peptide epitope.

[0069] Such second capture molecules can be used to increase thespecificity of the detection assay, as has already been described indetail in connection with the individual steps of the method.

[0070] It will be appreciated that the features and advantages whichhave been mentioned above and which will be explained hereinafter can beused not only in the stated combination but also alone or in anothercombination without leaving the scope of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0071] The basic principle of the method for the quantitative and/orqualitative analysis of proteins consists of employing an array of firstcapture molecules which are specific for peptide epitopes, where thereis detection with the protein-specific capture molecules immobilized inthe array format, not of proteins but of individual peptide fragmentscorresponding to the peptide epitopes after, for example, enzymaticcleavage of the proteins.

[0072] For this purpose, first potential peptide epitopes or proteinsequences are predicted theoretically in protein or gene sequencedatabases. Since individual epitopes may be split during the enzymaticor chemical cleavage of the proteins to be detected, account must betaken of the cleavage specificity of the proteases used for the proteindegradation in order to predict potential peptide epitopes. Potentialpeptide epitopes which have been predicted theoretically for theindividual proteins according to these criteria are prepared by chemicalpeptide synthesis and labeled on specific functional groups, for examplethe amino function of the N-terminus or the ε-amino group of lysine, forexample with fluorophore, biotin etc.

[0073] These peptide epitopes then are used for the production, knownper se, of antibodies by immunization. The capture molecules produced inthis way, which may in the simplest case be antibodies, are thenimmobilized in rows and columns on the array.

[0074] Protein fragments corresponding to the peptide epitopes areobtained by enzymatic or chemical degradation reaction of all theproteins in the entire protein mixture to be analyzed, using specificproteases such as, for example, trypsin, endoprotease Lys C etc., andare then incubated with the array and are detected on the basis of theirbinding to the first capture molecules.

[0075] For detection, it is possible on the one hand to label theresulting peptide fragments with a marker such as, for example,fluorophore, biotin etc., at the same specific functional groups as thesynthetic peptide epitopes employed to generate the specific capturemolecules. The correspondingly labeled peptides are incubated on theantibody arrays, the unbound peptide fragments are washed away, and thebound peptide fragments are detected site-specifically via theirlabeling. Since complete labeling of the peptides is possible, eachbound peptide epitope can be determined quantitatively.

[0076] On the other hand, it is also possible to detect unlabeledpeptide epitopes if a labeled second capture molecule is employed, whichmay in the simplest case be a second antibody which recognizes thepeptide bound to the immobilized antibody. This second antibody isgenerated against the peptide bound on the immobilized antibody,resulting in a drastic increase in the selectivity, because only thespecific peptide fragment on the immobilized first antibody isrecognized, and peptide fragments nonspecifically bound there are notbound by the second antibody.

[0077] In this way, only peptides which represent potential immunogensand which are not fragmented in the protein degradation are employed forthe immunization, which leads to a saving of chemical peptide synthesisand the preparation of specific capture molecules, becausehigh-affinity, peptide fragment-specific capture molecules can beproduced with a high success rate. This leads to fewer capture moleculerejects than in the preparation of protein-specific capture molecules.

[0078] The binding specificity of the capture molecules obtained, inthis case therefore antibodies in the array format, can be determined bya binding assay with defined synthetic peptides which have been alteredin the individual amino acids. An alternative possibility is also toincubate synthetic peptide libraries which comprise all theoreticallypossible peptide sequences with individual antibodies immobilized forexample on affinity chromatography columns. The peptides bound from thepeptide mixtures can be eluted after denaturation of the antibody and beidentified by mass spectrometry or by Edman degradation.

[0079] It is possible with the novel method for proteins from thedegradation of cells, from body fluids or tissues not only to bequantified but, for example, also to be analyzed for post-translationalmodifications and single nucleotide polymorphisms.

What is claimed is:
 1. A method for analyzing a protein, comprising:preparing a first capture molecule that is specific for a peptideepitope; immobilizing said first capture molecule on an array; digestingthe protein to be analyzed to peptide fragments wherein at least one ofthe fragments corresponds to the peptide epitope; incubating said firstcapture molecule immobilized on the array with the peptide fragments;whereby at least one of said fragments binds to said first capturemolecule; and detecting said peptide fragment bound to the first capturemolecule.
 2. The method of claim 1, further comprising washing awayunbound peptide fragments after the incubating step.
 3. The method ofclaim 2, further comprising labeling said peptide fragments before theincubating step.
 4. The method of claim 3, wherein the first capturemolecule is generated to be specific for the peptide epitope, saidpeptide epitope is prepared well-aimed in relation to the peptidefragment resulted from digestion of the protein to be analyzed.
 5. Themethod of claim 3, wherein the first capture molecule is selected fromthe group consisting of: an antibody, an antibody fragment, a peptideaptamer and another capture molecule which can be prepared recombinantlyby mutation of binding domains and by chemical synthesis.
 6. The methodof claim 2, wherein said detecting is performed via a labeled secondcapture molecule which specifically recognizes the peptide fragmentbound to the first capture molecule.
 7. The method of claim 6, whereinthe second capture molecule is generated to be specific for a complex ofsaid first capture molecule and said peptide epitope bound thereto. 8.The method of claim 2, wherein the first capture molecule is generatedto be specific for a peptide epitope, said peptide epitope is preparedwell-aimed in relation to the peptide fragment resulted from digestionof the protein to be analyzed.
 9. The method of claim 2, wherein thefirst capture molecule is selected from the group consisting of: anantibody, an antibody fragment, a peptide aptamer and another capturemolecule which can be prepared recombinantly by mutation of bindingdomains or chemical synthesis.
 10. The method of in claim 1, furthercomprising labeling said peptide fragments before the incubating step.11. The method of claim 10, wherein said detecting is performed via alabeled second capture molecule which specifically recognizes thepeptide fragment bound to the first capture molecule.
 12. The method ofclaim 11, wherein the second capture molecule is generated to bespecific for a complex of said first capture molecule and said peptideepitope bound thereto.
 13. The method of claim 10, wherein the firstcapture molecule is generated to be specific for a peptide epitope, saidepitope is prepared well-aimed in relation to the peptide fragmentresulted from digesting of the protein to be analyzed.
 14. The method ofclaim 10, wherein the first capture molecule is selected from the groupconsisting of: an antibody, an antibody fragment, a peptide aptamer andanother capture molecule which can be prepared recombinantly by mutationof binding domains or chemical synthesis.
 15. The method of claim 1,wherein said detecting is performed via a labeled second capturemolecule which specifically recognizes the peptide fragment bound to thefirst capture molecule.
 16. The method of claim 15, wherein the secondcapture molecule is generated to be specific for a complex of said firstcapture molecule and said peptide epitope bound thereto.
 17. The methodof claim 16, wherein the second capture molecule is selected from thegroup consisting of: an antibody, an antibody fragment, a peptideaptamer and another capture molecule which can be prepared recombinantlyby mutation of binding domains and by chemical synthesis.
 18. The methodof claim 15, wherein the second capture molecule is selected from thegroup consisting of: an antibody, an antibody fragment, a peptideaptamer and another capture molecule which can be prepared recombinantlyby mutation of binding domains and by chemical synthesis.
 19. The methodof claim 18, wherein the binding specificity of the second capturemolecule is established by binding assays using peptides which have beenvaried in individual amino acids or their length.
 20. The method ofclaim 1, wherein the first capture molecule is generated to be specificfor a peptide epitope which is prepared well-aimed in relation to thepeptide fragment resulted from digesting of the protein to be analyzed.21. The method of claim 20, wherein the well-aimed prepared peptideepitope is selected from potential peptide epitopes of the protein to beanalyzed.
 22. The method of claim 21, wherein the potential peptideepitopes are derived from a known amino acid sequence of the protein tobe analyzed.
 23. The method of claim 22, wherein sequence sections whichare retained after digesting of the protein to be analyzed are selectedas potential peptide epitopes.
 24. The method of claim 21, wherein thepotential peptide epitopes are derived from a known nucleic acidsequence of the corresponding protein to be analyzed.
 25. The method ofclaim 21, wherein the sequence sections which are retained afterdigesting of the protein to be analyzed are selected as potentialpeptide epitopes.
 26. The method as claimed in claim 20, wherein thewell-aimed prepared peptide epitopes are labeled, modified, or both,like the peptide fragments after or during digesting of the proteins tobe analyzed.
 27. The method as claimed in claim 20, wherein thewell-aimed prepared peptide epitopes are provided withpost-translational modifications.
 28. The method as claimed in claim 20,wherein the well-aimed prepared peptide epitopes have at least one aminoacid exchange, a deletion, or a combination thereof of an amino acidcompared with the potential peptide epitopes.
 29. The method as claimedin claim 1, wherein the first capture molecule is selected from thegroup consisting of: an antibody, an antibody fragment, a peptideaptamer and another capture molecule which can be prepared recombinantlyby mutation of binding domains and by chemical synthesis.
 30. The methodas claimed in claim 29, wherein the binding specificity of the firstcapture molecule is established by binding assays using peptides whichhave been varied in individual amino acids or their length.
 31. Acapture molecule, which binds specifically to peptide epitopes whichcorrespond to peptide fragments into which proteins to be analyzed canbe digested.
 32. The capture molecule as claimed in claim 31, whereinthe peptide epitopes in the protein itself are not accessible to thecapture molecule.
 33. A capture molecule which binds specifically to acomplex of a capture molecule as claimed in claim 31 and thecorresponding peptide epitope.
 34. A capture molecule which bindsspecifically to a complex of a capture molecule as claimed in claim 32and the corresponding peptide epitope.